Biochemical and immunological methods will be utilized to study three acrosomal enzymes; namely acrosin, arylsulfatase and sperm neuraminidase. Acrosomal enzymes will be extracted and purified by molecular sieve, ion exchange and affinity chromatography from both rabbit and boar epididymal spermatozoa. The antigenicity of these proteins will be compared under iso- and hetero-immunization regimes. During the immunization period, levels of immunoglobulins (IgM, IgG and IgA) will be quantitated in serum and reproductive tract fluids in response to the specific enzymes and their mode of immunization. To quantitate immunoglobulin levels a solid-phase immunoassay, that allows detection in the nanogram range, will be developed. Purified enzymes will be studied to determine what effect they may have on the modification or removal or investments from homologous and heterologous pre- and post-ovulatory ova. Immunospecific probes developed for each enzyme will be examined for their inhibition of enzymatic activity and their ability to inhibit investment modification in various mammalian ova. These probes will also permit the localization of acrosin, arylsulfatase and sperm neuraminidase within spermatozoa after acrosomal membrane modification by several chemical and physical methods. Localization studies will be performed at the light- and electron microscope levels to determine the precise location of these enzymes within the intact and disrupted acrosome of both capacitated and non-capacitated spermatozoa. To test the functional role of anti-acrosin, anti-arysulfatase and anti-sperm meuraminidase on their acrosomal enzyme counterparts during fertilization, the immunospecific probes will be evaluated for their effect on 1) sperm penetration in vitro and 2) sperm penetration in vivo. These studies should permit us to more critically examine the role of acrosin, arylsulfatase and sperm neuraminidase in mammalian fertilization.